Substrate Specifity Profiling of the Aspergillus fumigatus Proteolytic Secretome Reveals Consensus Motifs with Predominance of Ile/Leu and Phe/Tyr

The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis

The Serine-Proline Turn: A Novel Hydrogen-Bonded Template for Designing Peptidomimetics

Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds

An Enhanced β Turn in Water

Aiming to design short linear peptides featuring strong intramolecular hydrogen bonds in water, a series of tetrapeptides based on the sequence Ac-Ala-Pro-Ala-Ala-NH₂ containing all possible combinations of l- and d-amino acids was synthesized. A regiospecific combination of heterochiral residues (ddll or its mirror image lldd) can be used to increase turn formation and stability within short peptides in water

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation

Hemopressin Forms Self-Assembled Fibrillar Nanostructures under Physiologically Relevant Conditions

The nonapeptide hemopressin, which is derived from the α chain of hemoglobin, has been reported to exhibit inverse agonist activity against the CB1 receptor. Administration of this peptide in animal models led to decreased food intake and elicited hypotensive and antinociceptive effects. On the basis of hemopressin’s potential in therapeutic applications and the lack of a structure–activity relationship study in literature, we aimed to determine the conformational features of hemopressin under physiological conditions. We conducted transmission electron microscopy experiments of hemopressin, revealing that it self-assembles into fibrils under aqueous conditions at pH 7.4. Circular dichroism and nuclear magnetic resonance experiments indicate that the peptide adopts a mostly extended β-like structure, which may contribute to its self-assembly and fibril formation

Deconvolution of <i>AF</i> protease substrate consensus motifs.

<p>For each IQFP motif with five or more wells demonstrating proteolytic cleavage by <i>AF</i> secreted proteases (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021001#pone-0021001-t002" target="_blank">Table 2</a>), a representative well was selected for deconvolution and further study. Individual IQFP sequences were synthesized and fine substrate specificity was determined. Although cleavage of the Ser/Thr–Ile/Leu–Phe/Tyr motif was promiscuous (<i>A</i>), others exhibited clear amino acid preferences, particularly at the <i>Zaa</i> position (<i>B, D</i>). Data represent fluorescence fold change after 2 h incubation.</p

Substrate specificity of <i>AF</i> secreted proteases and human serum.

<p><i>A</i>. Cleavage of an IQFP library by <i>AF</i> H237 culture supernatant. Each square corresponds to a single well of a 96 well microplate. The brightness of a given square corresponds to its fluorescence intensity, quantified as fluorescence fold change, which in turn indicates the extent of cleavage of the IQFP probes in that well. Of the 512 wells in the library, 93 exhibited greater than 4-fold fluorescence enhancement upon incubation with <i>AF</i> culture supernatant. <i>B</i>. Cleavage of the same IQFP library by complement preserved normal human serum. C. Number of IQFP sequences distinctly cleaved by only <i>AF</i> culture supernatant, only human serum, or both. <i>D</i>. Graphical depiction of amino acid preferences at each variable position of the library sequences cleaved by <i>AF</i> secreted proteases. Isoleucine, leucine, phenylalanine, and tyrosine were predominant at each position. Amino acid preference data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021001#pone-0021001-t001" target="_blank">Table 1</a>.</p

IQFP motifs cleaved by <i>AF</i> secreted proteases.

<p>Motifs appearing at least five times among the 93 hits are shown. From each of the four motifs exhibiting the highest mean fold change fluorescence enhancement, a representative well not cleaved by human serum was selected for deconvolution and further analysis.</p